Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae

The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H⁺-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments.     

Visualization of Atg3 during Autophagosome Formation in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.     

Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.

The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.     

Grayanotoxin-XVIII and grayanoside B,a new a-nor-b-homo-ent-kaurene and its glucoside from Leucothoe grayana

From Leucothoe grayana a new grayanoid (diterpene) and its glucoside have been isolated. The structures of these new natural compounds have been established by chemical and spectroscopic means and by correlation with a known compound.     

Drug resistance and R plasmids in Bordetella bronchiseptica isolates from pigs

A total of 207 strains of Bordetella bronchiseptica isolated from pigs in 1978 and 1979 were tested for drug resistance and for the properties of their R plasmids. Apart from intrinsic resistance to spectinomycin, single (sulfadimethoxine), double (sulfadimethoxine and streptomycin), andt riple (sulfadimethoxine, streptomycin, and ampicillin) resistance were found in 54.1%, 1.0%, and 15.9% of the strains, respectively. All of the triple-resistance determinants were associated with mercury resistance and were conjugative. pBB1, one of these R plasmids, was identified as Fi- (F) and Spp- (no suppression of phage multiplication) type, and as a member of incompatability group IncP. The single- and double-resistance determinants were nonconjugative. pBB2, one of the double-resistance determinants, was mobilized by an R plasmid, RP4, with the high efficiency of 80% and at a frequency of 3.3% without cotransfer of RP4. The molecular weight of pBB1 and pBB2 was estimated at 36 X 10(6) and 13 X 10(6) daltons, respectively, by electron microscopy and agarose gel electrophoresis. pBB1 had five cleavage sites for EcoRI endonuclease, and four sites for HindIII. pBB2 had two EcoRI sites, one HindIII, and one BamHI site. Cells carrying pBB1 or pBB2 produced enzymic activity tha inactivated streptomycin in the presence of ATP.     

Overexpression and cosuppression of xylem‐related genes in an early xylem differentiation stage‐specific manner by the AtTED4 promoter

Tissue‐specific overexpression of useful genes, which we can design according to their cause‐and‐effect relationships, often gives valuable gain‐of‐function phenotypes. To develop genetic tools in woody biomass engineering, we produced a collection of Arabidopsis lines that possess chimeric genes of a promoter of an early xylem differentiation stage‐specific gene, Arabidopsis Tracheary Element Differentiation‐related 4 (AtTED4) and late xylem development‐associated genes, many of which are uncharacterized. The AtTED4 promoter directed the expected expression of transgenes in developing vascular tissues from young to mature stage. Of T2 lines examined, 42%, 49% and 9% were judged as lines with the nonrepeat type insertion, the simple repeat type insertion and the other repeat type insertion of transgenes. In 174 T3 lines, overexpression lines were confirmed for 37 genes, whereas only cosuppression lines were produced for eight genes. The AtTED4 promoter activity was high enough to overexpress a wide range of genes over wild‐type expression levels, even though the wild‐type expression is much higher than AtTED4 expression for several genes. As a typical example, we investigated phenotypes of pAtTED4::At5g60490 plants, in which both overexpression and cosuppression lines were included. Overexpression but not cosuppression lines showed accelerated xylem development, suggesting the positive role of At5g60490 in xylem development. Taken together, this study provides valuable results about behaviours of various genes expressed under an early xylem‐specific promoter and about usefulness of their lines as genetic tools in woody biomass engineering.     

Inhibitory Effects of Trientine, a Copper-Chelating Agent, on Induction of DNA Strand Breaks in Kidney Cells of Long-Evans Cinnamon (LEC) Rats.

The effects of treatment with trientine, a specific copper-chelating agent, on the accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the kidneys of LEC rats in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, renal copper contents did not increase and were maintained at the same levels as those in 4-week-old LEC rats. Estimation of the amounts of DNA single-strand breaks (SSBs) by comet assay showed that SSBs of DNA were induced in a substantial population of LEC rat renal cortex cells around 12 weeks of age and that the amounts of SSBs increased in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that SSBs of DNA in LEC rat kidney cells are induced prior to occurrence of clinical signs of hepatic injury and that treatment of LEC rats with trientine decreases the number of DNA strand breaks.     

Ablation of the 14‐3‐3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex

14‐3‐3 proteins are ubiquitously‐expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14‐3‐3epsilon and 14‐3‐3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14‐3‐3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14‐3‐3gamma‐deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14‐3‐3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time‐lapse live imaging of brain slices revealed that the ablation of the 14‐3‐3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14‐3‐3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14‐3‐3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14‐3‐3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600–614, 2016     

Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.